Use of lactobacillus for treatment of virus infections

ABSTRACT

The present technology relates to the use of at least one strain of probiotic bacteria selected from  Lactobacillus  for the manufacture of a pharmaceutical composition for the treatment and/or prevention of a virus infection.

FIELD OF THE INVENTION

The present invention relates to the use of at least one strain ofprobiotic bacteria selected from Lactobacillus for the manufacture of apharmaceutical composition for the treatment and/or prevention of avirus infection.

BACKGROUND ART

Probiotic bacteria are defined as live micro-organisms which whenadministered in adequate amounts beneficially affect the host.Lactobacilli and bifidobacteria are the most frequently used bacteria inprobiotic products. These bacteria are generally safe, as are probioticsbased on these organisms. The lack of pathogenicity extends across allage groups and to immunocompromised individuals. Intake of differentprobiotic bacteria has been shown to have clinical benefits in variousphysiologic or pathologic situations. The most clear cut effects havebeen shown in diarrhea caused by antibiotic therapy or rotavirusinfection. There are also studies showing positive clinical effects ininflammatory bowel diseases, atopic dermatitis and hypercholesterolemiaafter intake of probiotic bacteria. The mechanism, by which probioticbacteria contribute to these clinical improvements are not clear. Invitro human, as well as both in vivo and in vitro animal studies haveshown that different species of lactobacilli affects the innate andacquired immune system in many different ways. Clinical studies havemainly shown stimulation of the innate cellular immune system andenhancement of humoral immune responses to natural infections andsystemic or oral immunisation. Regarding effects of the innate immunesystem, increased phagocytic activity of polymorphonuclear cells (PMN)and increased NK cell tumor killing activity have been reported. To ourknowledge, there are no clinical studies showing effects on the specificcellular immune system after intake of probiotic bacteria.

According to the present invention the effects on the innate andacquired immune system following daily intake of lactobacilli or theGram-negative bacteria P. lundensis have been thoroughly investigated.Interestingly, it has been observed an activation of the specificcellular immune system in subjects receiving L. plantarum andindications of such in subjects receiving L. paracasei. Moreover,immunity-enhancing effects on the innate immune system, such asexpansion of the NKT cell population and increased phagocytic activitywere observed in subjects receiving different lactobacilli species.Intake of the Gram-negative bacteria P. lundensis had no effects,whatsoever, on the different immune parameters measured according to theexperiments described herein.

The development of antibiotic resistance and failures in varioustreatment of infections have risen an increased interesting inprobiotics as an alternative tool. There might be a need for a probioticfunctional food product targeting the common cold problem. It is clearin terms of the high number of incidence of cold infections every year.Traditionally foods with high levels of vitamin C have been taken to tryto reduce the incidence of common cold. On the market there are a numberof different products claiming some effect on the immune system.

The present application will aim to study if a probiotic functional foodproduct after regular administrations could affect the common coldsymptoms in a similar way and thus can be an alternative solution forthis problem in the general community.

SUMMARY OF THE INVENTION

An object of the present invention is the use of at least one strain ofprobiotic bacteria selected from Lactobacillus for the manufacture of apharmaceutical composition for the treatment and/or prevention of avirus infection.

Another object of the present invention is a method for treatment and/orprevention of a virus infection, wherein at least one strain ofprobiotic bacteria selected from Lactobacillus is administered to anindividual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the numbers of volunteers reporting any minor adversegastrointestinal effects during the trial.

FIG. 2 shows base line numbers (day 0) of different lymphocytes per mlblood (mean±(SEM))

FIG. 3 shows base line (day 0) percentages or GMFI (mean±(SEM)) oflymphocytes positive for different cell activation and memory markers.

FIG. 4. Subjects were randomly assigned to nine different study groups.The trial started with a wash out period of two weeks. Thereafter, theactive study period followed. During this period, the subjects consumedone dose of study product per day for 14 (L. plantarum Heal 19, L.fermentum, L. paracasei, L. gasseri, L. rhamnosus, P. lundensis groups)or 35 days (L. plantarum 299v and placebo group). Each dose contained10¹⁰ coloni forming units (CFU) (lactobacilli groups) or 10⁹ CFUbacteria (P. lundensis group).

FIG. 5. Percentages of lymphocytes expressing the activation phenotypesCD8CD25, CD8HLA-DR, CD4CD25 and CD4HLA-DR was analysed by flowcytometry.Group means (±SEM) based on individual ratios, day 14/day 0 and day35/day 0 (for L. plantarum and placebo group only) is shown.

FIG. 6. Percentages of lymphocytes expressing the memory phenotypesCD8CD45RO and CD4CD45RO was analysed by flowcytometry. Group means(±SEM) based on individual ratios, day 14/day 0 and day 35/day 0 (for L.plantarum and placebo group only) is shown.

FIG. 7. Percentages of lymphocytes positive for the NKT cell markers(CD56CD16CD3) was analysed by flowcytometry. Group calculations arebased on individual ratios (day 14/day 0).

FIG. 8. The phagocytic activity of neutrophils was analysed byincubating whole blood cells with FITC-labelled E. coli or S. aureus.The ratio between mean fluorescence values obtained at day 14 and day 0was determined individually and group calculations are shown in thisfigure.

FIG. 9 shows the ratio of lymphocytes expressing the activationphenotypes CD4CD25 from experiment 2.

FIG. 10 shows the ratio of lymphocytes expressing the activationphenotypes CD4⁺CD25⁺⁺ from experiment 2.

FIG. 11 shows the ratio of lymphocytes expressing the activationphenotypes CD8⁺HLA-DR⁺ from experiment 2.

FIG. 12 shows the ratio of lymphocytes expressing the activationphenotypes CD8+CD25+ from experiment 2.

FIG. 13 shows the ratio of lymphocytes expressing the activationphenotypes CD4CD45RO from experiment 2.

DETAILED DESCRIPTION OF THE INVENTION

The Lactobacillus used according to the invention may be selected from,but not limited to, the group consisting of Lactobacillus plantarum,Lactobacillus rhamnsosus, Lactobacillus fermentum, Lactobacillusparacasei and Lactobacillus gasseri.

The Lactobacillus plantarum used according to the invention may beselected from, but not limited to, the group consisting of Lactobacillusplantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843,Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL19, DSM 15313, and Lactobacillus plantarum HEAL 99, DSM 15316.

The Lactobacillus paracasei used according to the invention may beselected from, but not limited to, the group consisting of Lactobacillusparacasei 8700:2, DSM 13434, and Lactobacillus paracasei 02A, DSM13432.The Lactobacillus gasseri used according to the invention may beselected from, but not limited to, Lactobacillus gasseri VPG44, DSM16737.

Other probiotic bacterial strains, than the ones explicitly disclosedherein, may naturally be used according to the present invention and arewithin the scope of the invention as long as they provide the desiredeffects, i.e. have a preventive effect on a virus infection oralleviates a virus infection.

In an embodiment of the invention at least two strains of probioticbacteria are used in the pharmaceutical composition. Said at least twostrains are intended to be administered sequentially or simultaneously.Thus, the strains may be administered in a mixture in one composition orthey may be administered in a sequence separately in differentcompositions.

According to the invention it is possible to treat virus infections.Treatable virus infections are i.e. those caused by a virus selectedfrom, but not limited to, the group consisting of herpes simplex Ivirus, herpes simplex II virus, herpes zoster virus, common cold virus,rhinovirus, adenovirus, parainfluenza virus, respiratory syncytialvirus, enterovirus and coronavirus. Any other virus infection, notspecifically mentioned here, that the probiotic bacteria have an effecton are also within the scope of the present invention. It is known thatthere are many different viruses and forms thereof that cause a commoncold. All such viruses are within the scope of the present invention.

In the present context the word “treatment and/or prevention” includes aprophylactic treatment of an individual, i.e. the treatment with theprobiotic bacteria is started before the disease or virus infection hasdeveloped in order to prevent the disease/infection, as well as atreatment of a disease/infection that already has developed in anindividual. In the latter case an alleviation of the symptoms is forinstance expected or the general condition of the patient is enhanced orthe patient is cured from the disease/infection faster. Thus, theindividual may be a person at risk for developing an infection or not oran infection has already developed in the patient.

In an embodiment of the invention each of said strain(s) is present inthe pharmaceutical composition in an amount of, but not limited to,about 1×10⁶ to about 1×10¹⁴ CFU, preferably from about 1×10⁸ to about1×10¹², and more preferably from about 1×10⁹ to about 1×10¹¹.

The pharmaceutical composition according to the invention may e.g. be aliquid formulation or a solid formulation.

When the pharmaceutical composition is a solid formulation it may beformulated as a tablet, a sucking tablet, a sweet, a chewing tablet, achewing gum, a capsule, a sachet, a powder, a granule, a coatedparticle, a coated tablet, an enterocoated tablet, an enterocoatedcapsule, a melting strip or a film.

When the pharmaceutical composition is a liquid formulation it may beformulated as an oral solution, a suspension, an emulsion or syrup. Saidcomposition may further comprise a carrier material independentlyselected from, but not limited to, the group consisting of oat mealgruel, lactic acid fermented foods, resistant starch, dietary fibres,carbohydrates, proteins, and glycosylated proteins.

In an embodiment of the invention said pharmaceutical composition is amedical food, a functional food, a dietary supplement, a nutritionalproduct or a food preparation.

The pharmaceutical composition according to the invention, usedaccording to the invention or produced according to the invention mayalso comprise other substances, such as an inert vehicle, orpharmaceutical acceptable adjuvants, carriers, preservatives etc., whichare well known to persons skilled in the art.

The wording “pharmaceutical composition” does not necessarily need to bea pharmaceutical composition in its normal sense but may be formulatedas a food composition, a dietary supplement, a functional food, amedical food or a nutritional product as long as the required effect isachieved, i.e. treatment or prevention of virus infections. Said foodcomposition may be chosen from the group consisting of beverages,yoghurts, juices, ice creams, breads, biscuits, cereals, health bars,spreads and nutritional products. The food composition may furthercomprise a carrier material, wherein said carrier material is chosenfrom the group consisting of oat meal gruel, lactic acid fermentedfoods, resistant starch, dietary fibres, carbohydrates, proteins andglycosylated proteins.

Thus, the use of a composition according to the invention may be verybeneficial in the sense of being usable prophylactically, i.e. beforethe virus infection has developed. Since the pharmaceutical compositionused is not necessarily a pharmaceutical composition in its normalsense, but can also be a dietary supplement or functional food, it isvery convenient for a normal healthy individual to take to compositionof the invention prophylactically.

EXAMPLES Example 1 Subjects and Trial Criteria

Fifty-seven apparently healthy volunteers within the age range 18-55years (median, 26 years) were selected for this blind placebo controlledstudy. Subjects were randomly assigned to eight groups, receiving eitherone of the following Gram-positive bacteria, L. plantarum 299v (n=7), L.plantarum Heal 19 (n=7), L. fermentum 35D (n=7), L. paracasei 8700:2(n=7), L. gasseri VPG44 (n=7), L. rhamnosus 271 (n=7), or theGram-negative bacteria, P. lundensis (n=7) or placebo (n=10). The doseof bacteria was 10¹⁰ bacteria/day for lactobacilli and 10⁹ bacteria/dayfor P. lundensis. The control group took skim milk powder (1 g).Depending on the group, the study had a duration period of 6 or 9 weeksconsisting of two weeks wash out period, 2 or 5 weeks active studyperiod and 2 weeks follow up period (FIG. 4). Each subject was suppliedwith a list of products containing probiotic products, which should notbe consumed during the whole study period. Peripheral blood samples werewithdrawn from subjects by venipuncture at two or three time points, day0, day 14 and day 35. A diary, in which each subject stated adverseeffects, health conditions and confirmed intake of study product, waskept during the trial.

Flow Cytometry

Phenotypic analysis of lymphocytes in whole blood was performed by flowcytometry. The following anti-human monoclonal antibodies were used assurface markers for different cell populations: CD3 FITC (SK7), CD4 APC(SK3), CD8 PerCP (SK1), CD19 PerCP (SJ25C1), CD56 PE (MY31), CD16 PE(B73.1), and CD5 FITC (L17F12). Following anti-human monoclonalantibodies were used for detection of different activation and memorymarkers: CD25 FITC (2A3), HLA-DR PE (L243), CD45RO PE (UCHL-1), CD38 PE(HB7), CD27 PE (L128), and CD11b PE (D12). All antibodies were purchasedfrom Becton-Dickinson (Erembodegum, Belgium). Whole blood (100 μl) wasincubated with antibodies (10 μl/antibody) for 30 min at 4° C. in thedark. Thereafter, 2 ml of FACS lysing solution (Becton-Dickinson) wasadded and incubated for 15 min at 20° C. in the dark. Cells were washedby adding 3 ml FACSFlow and centrifuged at 300×g for 5 min. Washed cellswere resuspended in 200 μl FACSFlow and analysed on a FacsCalibur(Becton-Dickinson) with CellQuest software.

Phagocytosis Assay

The phagocytic activity of granulocytes and monocytes were quantifiedwith PHAGOTEST™ (Orpegen Pharma, Heidelberg, Germany) according tomanufacturers instruction with some modifications. Briefly, 20×10⁶ FITClabelled E. coli or FITC labelled S. aureus was added to pre-cooledwhole blood (100 μl). Blood cells and bacteria were incubated on 37° C.for 10 FacsCalibur with CellQuest software.

Calculations

Individual changes regarding different immune parameters were determinedby calculating the ratio between the individual values obtained at day14 and day 0, or the values at day 35 and day 0. These ratios were usedfor all group calculations and statistics.

Statistics

All statistical analyses were performed using Statview. Mann-Whitney Utest were used to compare different groups.

Results Clinical Observations

Fifty-four out of fifty-seven volunteers completed the study. Twopersons were excluded due to infection and antibiotic treatment (one inthe placebo group and one in the group receiving P. lundensis). Oneperson was excluded day 16 due to pregnancy (placebo group). Only mildadverse gastrointestinal side effects were reported following intake ofstudy products (FIG. 1).

Intake of Lactobacilli Activates T Cells

There were great baseline (day 0) individual variations regardingactivation markers on CD4⁺ and CD8⁺ T cells. The baseline percentages ofcells expressing different cell surface markers are shown in FIG. 2. Nosignificant differences were observed between different groups at thistime point. Since huge inter-individual variations were observed, it waschosen to compare ratio values at day 14 and day 35 compared to day 0for each individual. All calculations and comparisons were done on theseratio values (day 14/day 0 and day 35/day 0). After 14 days of intake ofstudy product containing L. plantarum 299v there was an approximatelytwofold increase of the expression of the activation marker CD25 on CD8⁺T cells (p=0.01) (FIG. 5). There was also a strong, although notsignificant (p=0.12), indication of upregulation of HLA-DR on CD8⁺ cellsfollowing L. plantarum 299v intake. In addition, it was also observed atendency towards activation of CD4⁺ T cells after L. plantarum 299vintake. Intake of the other lactobacilli species included in this study,as well as the Gram-negative bacteria P. lundensis activated neitherCD8⁺ nor CD4⁺ T cells. However, there was a tendency that intake of L.paracasei did increase the expression of HLA-DR on CD4⁺ T cells(p=0.18).

Intake of Lactobacilli Induces a Memory Phenotype of CD4⁺ T Cells

Geometric means of the fluorescence intensity (GMFI) of the expressionof CD45RO on CD4+ and CD8+ T cells were compared between groupsreceiving different study products. As above, group calculations basedon individual ratio values (day 14/day 0 and day 35/day 0) were used forcomparisons. After 35 days of intake of study product containing L.plantarum 299v the CD45RO GMFI on CD4+ T cells increased significantly(p=0.03). There was also a tendency towards increased CD45RO expressionon CD8+ T cells following intake of L. plantarum (FIG. 6). Moreover,intake of L. paracasei seems to have a positive effect on upregulationof CD45RO on CD8+ T cells (p=0.10) (FIG. 6).

Effect on Different Cell Populations Following Intake of Study Product

Following intake of L. pararcasei there was an increase in thepercentage of lymphocytes being identified as NKT cells (P=0.06) (FIG.7). Relative increase/decrease compared to day 0 could not be detectedregarding other cell populations, such as CD4+ T cells, CD8+ T cells, Bcells, B-1 cells (CD19+CD5+), NK cells, granulocytes and monocytes.

Phagocytic Activity

Granulocytes and monocytes were identified in the FSC-SSC diagram. Theability of these cells to phagocytose FITC-labelled Gram-positive orGram-negative bacteria was tested. As shown in FIG. 8, granulocytes fromvolunteers given L. plantarum 299v (p=0.064), L. plantarum Heal 19(p=0.064), L. fermentum (p=0.064) or L. paracasei (p=0.05) were moreefficient then granulocytes from placebo treated volunteers inphagocytosis of the Gram-negative bacteria E. coli. However, there wasno difference between the groups in phagocytosis of the Gram-positivebacteria S. aureus. No differences in the phagocytic activity ofmonocytes could be detected (data not shown).

Discussion

The primary task of the immune system is to react rapidly and violentlyto micro-organisms thereby preventing and curing infections. The killingof microorganisms employs powerful mechanisms that also cause harm toour own tissues. Therefore, it is necessary that it neither reacts toour own tissues, nor to innocuous substances present in the environment.Therefore, the immune system develops and maintains tolerance both tothe components of our own body, and to food and inhaled proteins. Ifthis fails, a number of diseases may arise. Means to develop specificimmune tolerance are an essential task of the immune system.

A central role in all immune reactions is played by the T helper cell.When a T helper cell becomes activated by its specific antigen, itbecomes activated, divides, matures and produces a range of cytokineswhich direct the action of other types of cells in the immune system,such as cytotoxic T cells and B cells. Activation of T helper cells isnecessary in order to produce most types of immune reactions, includingproduction of antibodies. Conversely, if activation of T helper cells isprevented, most types of immune reactions are paralysed.

There are several mechanisms by which activation of T helper cells andmaintenance of tolerance is ensured. One mechanism is elimination in thethymus of T cells with capacity to recognize and react to own tissue.However, this elimination is not complete and, furthermore, we also needto develop specific immune tolerance to exogenous antigens. Otherwise wewould react violently to all types of inhaled and ingested substances,leading to massive inflammation and wasted immune resources.

A cell type that is central for maintenance of tolerance is theregulatory T cell. This cell type can be recognized by certain markers,such as surface expression of CD4 and CD25, possession of intracellularCTLA-4, and transcription of the nuclear protein Foxp3. The regulatory Tcells are capable of preventing other T cells to become activated whenencountering harmless substances and, hence, prevent all types ofunwanted immune reactions.

In the present context the symbol “+” in connection with a certainmarker such as CD4+ and CD25+ means that the marker is expressed on a Tcell. For instance CD4+CD25+ T cells are T cells that express both theCD4 marker and CD25 marker on its surface. However, nothing is saidabout the amount of the marker that is expressed, only that it ispresent. In the present context the symbol “++” in connection with amarker such as CD4++ or CD25++ means that there is a lot of markerexpressed. The regulatory T cells are those cells with a lot of CD25 onthe surface, i.e. CD4+CD25++ cells. On the other hand, CD4+CD25+ T cellsare only activated T cells. Sometimes the specific symbols “+” and “++”are not used, e.g. CD4CD25 only, and this means that the cells areactivated such CD4+CD25+ cells. Thus, CD4CD25 is the same as CD4+CD25+.When discussing regulatory T cells, it is always written as CD4+CD25++cells.

This blind placebo-controlled study is unique in that it is the firststudy comparing the influence of several immune parameters followingintake of different Gram-positive lactobacilli or the Gram-negativebacteria P. lundensis. Interestingly, intake of P. lundensis did notinfluence any of the measured parameters. In contrast, intake oflactobacilli affected different components of both the specific andinnate immune system. A novel finding in this study was that intake ofL. plantarum had a pronounced positive effect on activation andinduction of memory cells in the T cell populations. There was asignificant upregulation of the IL-2 receptor α chain (CD25) and astrong tendency towards upregulation of HLA-DR on cytotoxic T cells. Atendency towards upregulation of these activation markers was alsoobserved on helper T cells after intake of L. plantarum. Expression ofactivation markers indicates that the T cells have started toproliferate in response to antigen-specific or non-specific stimuli andthat these cells more readily exert their effector functions compared toresting T cells. The mechanisms behind L. plantarum induced activationof T cells could be via antigen presenting cells that are activated bytoll-like receptors binding to microbial compounds. Activation ofantigen presenting cells makes them more efficient in presenting antigento T cells. In addition, both helper and cytotoxic T cells have shown tohave various expressions of toll-like receptors, which probably makethese cells sensible for non-specific activation by microbial componentsand products.

In analogy to the helper T cell compartment, expression of CD45RO seemsto mark a memory population also among cytotoxic T cells. There wasfound a significant increase in the expression of this memory cellmarker on helper T cells, and a tendency towards upregulation oncytotoxic T cells following 35 days intake of L. plantarum. In addition,intake of L. paracasei also showed a tendency towards upregulation ofCD45RO on cytotoxic T cells. Relative to naïve T cells, CD45RO+ T cellscan secrete a broad spectrum of cytokines. Moreover, CD45RO+ T cells canproliferate and produce IL-2 when the CD3-TCR complex is stimulatedunder suboptimal conditions, whereas naïve T cells require a strongCD3-TCR stimulus to carry out these functions. The formation of memory Tcells is important for induction of an efficient immune response afterinfection and vaccination.

The innate cellular immune system was also affected by intake ofprobiotic bacteria. It was demonstrated that the natural killer T (NKT)cell population was expanded following intake of L. paracasei. NKT cellsconstitute a lymphocyte subpopulation that coexpress the NK cell markerCD56 and the T cell marker CD3-T cell receptor complex. Studies in bothhumans and mice have demonstrated that NKT cells play a central role inthe regulation of autoimmune diseases, such as multiple sclerosis, typeI diabetes, and systemic lupus. NKT cells also exert effector functionsagainst tumour and virus infected cells. Thus, NKT cells are pleotropicin their functions. Other clinical studies evaluating the immunologicaleffects of probiotic bacteria have shown that intake of L. rhamnosusHN001 and Bifidobacterium lactis HN019 enhance NK (including NKT) celltumour killing activity of K562 cells. In this study it was alsoconfirmed the observation by others that phagocytic activity ofpolymorphonuclear cells is increased after intake of differentlactobacilli. The consequence of the observed effects on the differentimmune parameters in the present study is that one could speculate thatthe coincident activation of cytotoxic T cells and NKT cell expansionpoints to a strengthened immune defense against viral infections and/ortumours. The in vitro finding that lactobacilli induce mononuclear cellsto secrete IL-12 and IL-18, supports the theory that intake of thesebacteria stimulates cell-mediated activity.

In accordance with the present invention it has been concluded thatintake of L. plantarum and L. paracasei has a profound effect on thespecific and innate cellular immune system. However, the increase inimmune function demonstrated herein is for the time being difficult tocorrelate to a proven health benefit in humans. In order to address thisspecific issue, further clinical trials in individuals suffering frome.g. viral infections or tumours need to be accomplished. In suchstudies, it would be of special interest to compare the effect ofadministration of L. plantarum and L. paracasei separately or incombination.

Example 2

The goal of this example was to investigate the effect on the immunesystem by giving the same species of lactobacilli for a longer period oftime compared to several lactobacilli (different species) administeredin a sequence one after the other.

The volunteers were given a powder with freeze-dried bacteria during 14or 35 days. As gram-positive bacteria the probiotic bacteriaLactobacillus plantarum 299v is used alone or in combination with L.rhamnosus, L. fermentum, L. paracasei, and L. gasseri. As gram-negativebacteria Pseudomonas lundensis is given.

The following groups are studied:1) Lactobacillus plantarum 35 days2) L. plantarum 7d, L. rhamnosus 7d, L. fermentum 7d, L. paracasei 7d,L. gasseri 7d. Totally 35 days. (Sequence)3) A mixture of L. plantarum, L. rhamnosus, L. fermentum, L. paracasei,L. gasseri. Totally 14 days4) L. rhamnosus 14 days5) L. fermentum 14 days6) L. paracasei 14 days7) L. gasseri 14 days8) Pseudomonas lundensis 14 daysControl group 1) Placebo 35 daysControl group 2) Placebo 14 days

Blood samples are taken at day 0, 14 and 35. The amount of helper Tcells (CD4+) expressing high amounts of CD25 was defined in each groupby flow cytometry as have been explained above in experiment 1.

Results

On day 14, there was a borderline significance of CD4+CD25++ T cellsbeing expanded in individuals consuming the sequence of five differentlactobacilli strains.

Discussion

T helper cells (CD4+) expressing high density of the CD25 molecule(CD4+CD25++) have been shown to be important in order to protect againstautoimmune diseases, allergies and inflammatory bowel diseases. Thefinding that these cells are expanded after intake of a sequence ofdifferent lactobacilli indicate that intake of these bacteria might bebeneficial for the individual concerning the risk of developing theabove mentioned diseases.

Experiment 3

The aim of the present study is to investigate whether intake of thelactic acid bacteria in a freeze-dried formula/functional food productduring at least 3 months influences the severity of symptoms and theincidence and duration of common cold.

It is important that this is carried out in vivo in humans, as neitherin vitro studies nor animal studies would reflect the degree of efficacywhen administered to humans. The ability of these bacteria to becomeestablished in the intestine when administered directly aftercultivation is documented in earlier studies.

Thus, the objective is to investigate if consumption of a mixture ofLactobacillus plantarum 299v (DSM 9843) and Lactobacillus paracasei8700:2 (DSM 13434) (1×10⁹ cfu/d) can reduce the risk for common cold.

The study will take place during 90 days and 500 individuals will takepart in the study. 250 individuals will receive the active product and250 individuals will be given a placebo.

The study will be randomized, double blind and placebo-controlled withtwo parallel arms.

Exclusion criteria are as follows: Known intolerance or allergy to anyingredient included in the formulations; allergy medically treated;Current treatment for severe gastrointestinal disorders; Pregnancy orlactation; Vaccination against influenza within the last 12 months; andsmokers.

The probiotic given: Lyophilized Lactobacillus plantarum 299v andLactobacillus paracasei 8700; 2. Sucrose, maltodextrine and hydrolysedgelatine are added as cryoprotectants. Dosage will be a daily intake of1 g lyophilized Lactobacilli (approximately 1×10⁹ cfu/day). The dose istaken in association with breakfast.

The products will be produced, packed and labelled by Probi AB, Lund,Sweden. The quality of the product will also be checked by Probi AB.Each sachet will be labelled with the name of the study, the best bydate, how they are to be stored, the name of the manufacturer, the nameof the responsible investigator and her/his telephone number. Inaddition to the above information, a number denoting the subject isadded on the secondary package. A detailed instruction for dissolutionand intake will be inserted in the secondary package. The product willbe supplied in sachets.

From day—14 to day 104, the subject may not ingest products containingprobiotic bacteria. The subject will be provided with a list ofprobiotic products not allowed to be consumed during the study period.

Faecal samples are to be handed in on days 1 (before intake of studyproduct), 15 (after intake), and 104 (after intake). The samples shouldbe collected in two tubes no more than 18 hours before being handed infor analysis, and during this period are to be stored in a refrigerator.The samples will be analysed for lactobacilli.

Blood samples are to be taken day 1 and 15. The samples will be analyzedfor CD4+ and CD8+.

In view of experiments 1 and 2 it is expected that an enhancedprotection against common cold will be seen in the individuals takingthe probiotic mixture compared to the placebo group.

1. A method for preparing a pharmaceutical composition for the treatmentand/or prevention of a virus infection selected from the common coldvirus, rhinovirus, adenovirus, parainfluenza virus, respiratorysyncytial virus, enterovirus, and corona virus, comprising incorporatingat least one strain of probiotic bacteria of Lactobacillus selected fromthe group consisting of Lactobacillus plantarum 299, DSM 6595,Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9,DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, Lactobacillusplantarum HEAL 99, DSM 15316, Lactobacillus paracasei 8700:2, DSM 13434,and Lactobacillus paracasei 02A, DSM 13432 into the pharmaceuticalcomposition.
 2. The method of claim 1, wherein at least two strains ofprobiotic bacteria are used.
 3. The method of claim 2, wherein said atleast two strains are intended to be administered sequentially orsimultaneously.
 4. The method of claim 1, wherein said pharmaceuticalcomposition is a liquid formulation or a solid formulation.
 5. Themethod of claim 4, wherein said solid formulation is chosen from thegroup consisting of tablets, sucking tablets, sweets, chewing tablets,chewing gums, capsules, sachets, powders, granules, coated particles andcoated tablets, enterocoated tablets and capsules, and melting stripsand films.
 6. The method of claim 4, wherein said liquid formulation isselected from the group consisting of oral solutions, suspensions,emulsions and syrups.
 7. The method of claim 1, wherein said compositioncomprises a carrier material.
 8. The method of claim 1, wherein saidpharmaceutical composition is a medical food, a functional food, adietary supplement, a nutritional product or a food preparation.
 9. Themethod of claim 7, wherein said carrier material is selected from thegroup consisting of oat meal gruel, lactic acid fermented foods,resistant starch, dietary fibres, carbohydrates, proteins, andglycosylated proteins.
 10. The method of claim 8, wherein said foodpreparation is selected from the group consisting of beverages,yoghurts, juices, ice creams, breads, biscuits, cereals, health bars,and spreads.
 11. The method of claim 1, wherein each of said strain(s)is present in the composition in an amount of about 1×10⁶ to about1×10¹⁴ CFU.
 12. A method for the treatment and/or prevention of a virusinfection chosen from common cold virus, rhinovirus, adenovirus,parainfluenza virus, respiratory syncytial cirus, enterovirus and coronavirus, comprising administering to an individual a sufficient amount ofat least one strain of probiotic bacteria of Lactobacillus selected fromthe group consisting of Lactobacillus plantarum 299, DSM 6595,Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9,DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, Lactobacillusplantarum HEAL 99, DSM 15316, Lactobacillus paracasei 8700:2, DSM 13434,and Lactobacillus paracasei 02A, DSM
 13432. 13. The method of claim 12,wherein at least two strains of probiotic bacteria are administered. 14.The method of claim 13, wherein said strains are administered in asequence.
 15. The method of claim 8, wherein said carrier material isselected from the group consisting of oat meal gruel, lactic acidfermented foods, resistant starch, dietary fibres, carbohydrates,proteins, and glycosylated proteins.
 16. The method of claim 1, whereinsaid strain(s) is present in the composition in an amount of about 1×10⁸to about 1×10¹².
 17. The method of claim 1, wherein said strain(s) ispresent in the composition in an amount of about 1×10⁹ to about 1×10¹¹.18. The method of claim 13, wherein the strains are administeredsimultaneously.